Circadian rhythms and environmental lighting regulate a number of endocrine and behavioral functions. Arguably, the best understood endocrine rhythm is that of the pineal gland, which secretes the hormone melatonin almost entirely at night. Unlike cells from rat pineal, dispersed cells from chick pineal remain rhythmic in their synthesis of melatonin, and responsive to light, in culture. We undertook to identify the unknown photopigment that mediates photo-entrainment, the process by which light resets the endogenous clock, in collaboration with Mark Rollag and Ignacio Provencio (USUHS), and Maribeth Eiden (NIMH). We plan to use retroviral vectors carrying sense and antisense versions of candidate photopigments to test the effects of over- and under-expression of these proteins on the responses of chick pineal cells to light. Melanopsin and pinopsin are each novel photopigments present in chick pineal cells and the best candidates for the photopigments mediating photoentrainment. Iodopsin, a photopigment used for color vision in the retina, but absent from the pineal, provides a control. Last year, experiments transducing GFP into the cells were successful as proof of principle. This year, we isolated the cDNAs for melanopsin, pinopsin, and iodopsin, inserted them into plasmids, and prepared retroviral vectors containing these genes. We also determined the endogenous levels of gene expression for each photopigment gene and its tissue distribution using quantitative PCR. We optimized the experimental conditions for transduction and confirmed that the transduction process increases the expression of these genes considerably without harming the cells or itself affecting the circadian physiology of the cells. With the help of a commercial contractor we synthesized peptides from melanopsin and pinopsin and generated antibodies to assess the endogenous expression of the three proteins of interest and the effect of transduction on their expression. We have not quite completed validation of the specificity of these antibodies and optimization of their use. However, they have already shown that the endogenous tissue distribution of two of the three photopigment proteins matches the distribution of their endogenous mRNAs. We expect, in the coming year, to be able to follow the effects of transduction at the mRNA and protein levels and to proceed with the physiologic experiments indicated to determine the roles of these photopigment candidates in photoentrainment. We are also interested in the unknown signal transduction pathway from the photopigment to the clock. We undertook to determine whether activation or inhibition of MAPK pathways are necessary or sufficient for photoentrainment by examining the effects of a number of drugs. Last year we found that the effects of agents on levels of activated MAPK do not correlate with their effects on the circadian clock, as indicated by their ability to induce phase shifts. Drugs that inhibit MAPK activation, PD 98059 and U0126, and drugs that increase levels of activated MAPK, cycloheximide and chicken serum, were tested. One of each pair induced phase shifts and the other did not. Similarly, of two agents that induce phase shifts, high salt and caffeine, one lowered MAPK levels while the other had no effect. This year, we found a remarkable new effect: Changing the medium is sufficient to induce large (8-fold), rapid (peak at 5 minutes), and transient (return to normal in 2 hours) increases in levels of activated MAPK. Whether the medium is changed or not (at times that light and other agents induce phase shifts) made no difference to the phase of the melatonin rhythm in subsequent cycles. With appropriate controls, we also found that light pulses, which prominently induce phase shifts, had no effect on activated MAPK levels. Before concluding this project, however, to be sure that our measurements of activated MAPK levels have reflected events in the pinealocytes themselves, we are checking the proportion of cells in the culture that are indeed pinealocytes (using antibodies against the pinealocyte specific enzyme HIOMT) and the proportion of cells expressing MAPK (using antibodies against activated and total MAPK) that are pinealocytes. Preliminary results suggest that pinealocytes comprise the major cell type in the cultures and that MAPK is present in virtually all the cells. Taken together, the evidence indicates that changes in activation of MAPK are neither necessary nor sufficient for entrainment of the circadian clock in these cells.